Embodiments described herein generally relate to detection of circulating tumor cells.
Cancer remains a major public health issue in the United States as well as in other regions of the world. One in every four deaths in the United States can be attributed to one or more types of cancer. According to statistical data available from the National Center for Health Statistics and the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) Program for the years from 1930 to 2010, men have roughly a 44% chance of developing cancer in their lifetimes, as compared with a 38% chance for women. Early detection is the key to effective diagnosis, treatment and increased survival rate.
It is well known that the major cause of cancer-associated mortality (upwards of 90%) is tumor metastasis. Tumor metastasis is the spread of tumor cells from a primary tumor in a tissue of origin to a secondary tissue in the organism, by route of tumor cells which are dislodged or otherwise released from the tumor (e.g., circulating tumor cells or CTCs). CTCs are cells that have been released from the primary tumor and enter the vasculature or lymphatic system and circulate in the bloodstream. CTCs can affix themselves, through a variety of means, to the secondary tissue and grow in distant (anatomically speaking) locations, such as vital organs. CTCs have been detected in several epithelial cancers including breast, prostate, ovarian, lung and colon cancer. For women, the combination of breast, ovarian, lung and colon cancer equates to roughly 50% of the estimated new cancer cases that will result in death in the United States alone. For men, considering the combination of breast, prostate, lung and colon cancer, this number is just slightly lower at 46%. As such, detecting CTCs in the early stages of metastatic diseases would be very helpful relative to defining a well-timed and more effective therapy. As CTCs are directly related to cancer progression and are not detected in healthy patients or patients with non-malignant cancers, lower numbers of CTCs in the bloodstream equate to longer survival time and decreased severity of prognosis. The biggest challenge surrounding the detection of CTCs is the extremely low concentration in which these cells exist in the early stages of metastasis. In these early stages, CTCs can exist in extremely low concentrations (<10 CTCs/mL of blood). Further, it becomes very difficult to accurately detect CTCs in the presence of approximately 10 million leukocytes and approximately 5 billion hemocytes in 1 mL of whole blood.
To date, many assays have been developed to assist in the detection of CTCs in a patient's peripheral blood, which is blood obtained from circulation which is remote from the heart. One assay for the detection of CTCs called CELLSEARCH CTC, available from Janssen Diagnostics in Raritan, N.J. This system utilizes density gradient centrifugation to separate CTCs, based on the fact that CTCs are larger in size than leukocytes and hemocytes. The separation is followed by further purification processes including immunomagnetic cell enrichment using antibodies targeting an epithelial cell adhesion molecule (EpCAM), nucleus labeling with fluorescent dye and flow cytometry. One concern with such an assay lies in that of the filtration as filter edges can damage trapped cells, leading to lower cell viability. Another concern is possible loss of CTCs in the centrifugation step. Finally, the further purification relies on surface expression of EpCAM, which may be downregulated in some CTCs. All of the above deficiencies can lead to false negatives, especially in early detection. Additionally, methods such as that of CELLSEARCH CTC require large volumes of blood (7.5 mL), in an attempt to overcome the above noted deficiencies.
Conventional flow cytometry is a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by optical detection of fluorescence. The optical detection of flow cytometry is accomplished through alignment of cells into a single file line where a tightly focused laser is used to detect scattering and fluorescent light from individual cells. A Photo Multiplier Tube (PMT) is used to convert and amplify the photons emitted for detection by one or more fluorescence detectors. Although this method is very good in terms of accuracy and multi-parameter qualification, throughput is its largest shortfall. Limited throughput is especially important when taking into consideration a system for point-of-care usage, such as for use in a clinical setting. CTC viability is another of this method's drawbacks as well as capture efficiency. Further, low flow rates (in the vicinity of 1-2 mL/h) are standard requiring many hours for the completion of analysis.
Another implementation which helps to address the throughput issue faced by the Cell Search method is that of Fiber-optic Array Scanning Technology, or FAST. This assay claims detection of rare CTCs at a rate of nearly 500 times faster than that of ADM (Automated Digital Microscopy) with comparable sensitivity and improved specificity. In this method, a laser is used for improved exposure time, but the key innovation claimed by the FAST method is the use of an array of optical fibers that forms a wide collection aperture and 100 times field of view improvement over standard ADM.
Although a reduction in image resolution is suffered by the FAST assay, it remains adequate for the detection of fluorescently labeled cells. The large field of view is achieved by an array of optical fibers with asymmetric ends. The collected emission from the fiber array is subsequently collimated and then filtered using dichroic filters. The signal is then detected by a PMT. The focused beam from the laser is transformed into an elliptical spot via the galvanometer and F-Theta lens (in the absence of distortion, the position of the focused spot is dependent on the product of the Focal Length and the tangent of the deflection angle). Although this assay provides a significant increase in throughput over that of independent flow cytometry or the previously described Cell Search method, manual intervention requiring additional study of suspect cells using ADM remains necessary to differentiate false positives and/or negatives. Although the FAST assay provides a much faster method of CTC detection, the clinical setting can still benefit from further speed improvements and increases to both sensitivity and specificity in detection.
As such, there is a need in the art for devices, systems and methods to improve throughput and detection sensitivity and specificity in CTC detection.